2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.     

Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin

Naturally occurring CD4(+)CD25(+) regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4(+)CD25(+) non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4(+)CD25(high) T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4(+)CD25(low) or CD4(+)CD25(-) T cells did not. The expanding pure populations of CD4(+)CD25(high) T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4(+)CD25(-) T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4(+)CD25(high) T cell populations retained a broad TCR repertoire and, like CD4(+) CD25(+) T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4(+) and CD8(+) T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.     

Die Differenzierung isolierter Nerven- und Gliazellen aus trypsiniertem Rückenmark von Hühnerembryonen in Gewebekulturen

Zusammenfassung Cervikales und thorakales Rückenmark 6 und 7 Tage alter Hühnerembryonen wurde trypsiniert. Die isolierten Rückenmarkszellen wurden kultiviert und anschließend licht- und elektronenmikroskopisch untersucht.Unmittelbar nach der Isolierung kann man die Zellen morphologisch nicht unterscheiden. Nach wenigen Stunden der Inkubation beobachtet man ein erstes Auswachsen feiner Portsätze. Nach ca. 12stündiger Kulturzeit beginnt eine Zusammenlagerung der Zellen in Zellklumpen und lockeren Zellverbänden immer deutlicher zu werden, ohne daß eine aktive Zellbewegung sichtbar wird. Die Zusammenlagerung der Zellen geht mit einer feinstrukturell verfolgbaren Ausreifung einher.Am Ende der 1. Woche findet man neben einigen astrocytären Gliazellen auch zahlreiche andere polymorphe Formen, die sich weder als Astroglia- noch als Oligodendroglia-Zellen typisieren lassen. Letztere konnten während der kurzen Inkubationsdauer weder licht- noch elektronenmikroskopisch identifiziert werden.Die Nervenzellen zeigen eine Zunahme ihrer Organellen und eine Zusammenlagerung ihres endoplasmatischen Retikulums zu Ergastoplasmaformationen. Axone und Dendriten sind bereits nach 3tägiger Inkubation erkennbar.Ein eindeutiges Kriterium der Nervenzellausreifung stellt die Bildung von Synapsen dar, die nach dem 5. Tag der Inkubation deutlich wird. Es bilden sich axo-dendritische, axosomatische und axo-axonale Synapsen.Die mögliche physiologische Bedeutung der Synapsen auf Grund ihrer Ultrastruktur und die Bedeutung der 'membrane junctions wird diskutiert.

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On the differentiation of isolated nerve and glia cells from trypsinized spinal cord of chicken embryos cultivated in vitroA light- and electronmicroscopical investigation

Summary Cervical and thoracic segments of the spinal cord of six and seven days old chicken embryos were trypsinized. The isolated cells have been cultivated in vitro and studied both light- and electronmicroscopically.Immediately after isolation, neurons or glial cells can not be distinguished by morphological criteria. After a few hours in vitro, however, the first sprouting of fibres is visible. About 12 hours later an aggregation of cells in bulks or loosely woven nets becomes more and more prominent, without an active cellmovement visible. The aggregational process of the previously dissociated cells is combined with ultrastructural differentiation.At the end of the first week in vitro one could recognize besides some astrocytes a great number of those cells which were neither astroglia-nor oligodendroglia-cells. The latter were not detectable during our short periods of cultivation.Those cells showing an enhancement of organelles and their endoplasmic reticulum formed an ergastoplasm after five to seven days in vitro are definitely nerve cells, whose axons and dendrites appear after three days of incubation.The most convincing criteria of nerve cell maturation is the appearance of synapses, which increase in number from the fifth day of incubation onwards.Axo-dendritic, axo-somatic and axo-axonal synapses are present.A possible physiological significance of the synapses in relation to their ultrastructural features and the importance of the membrane junctions has been discussed.

Die Verff. danken Fräulein R. Dietrich und Frau M. C. Weinrichter für technische Assistenz.     

Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells

A limiting-dilution assay was developed and used to determine the frequency of autologous tumor-reactive cytotoxic T lymphocytes (CTL) in peripheral blood of a melanoma patient MZ2, who has been free of detectable disease since several years. In this patient, the frequencies of tumor-reactive CTL spontaneously varied only by a factor of 1.5. After vaccinations with autologous mutagenized and lethally irradiated tumor cells a two- to tenfold increase in frequencies of tumor-reactive CTL was found within the first 2 weeks. Thereafter, CTL frequencies returned to values measured prior to vaccinations. We conclude, that the limiting-dilution assay applied in this study can detect changes in the T cell response to autologous tumor cells. The frequency of tumor-reactive CTL determined with this approach can serve as an immunological parameter for monitoring the T cell response to autologous tumor cells in individual cancer patients receiving tumor cell vaccinations.     

The design and synthesis of diaryl ether second generation HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with enhanced potency versus key clinical mutations

Using a combination of traditional Medicinal Chemistry/SAR analysis, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad antiviral activity against a number of key clinical mutations.     

The crystal structure of shikimate dehydrogenase (AroE) reveals a unique NADPH binding mode

Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-A resolution and its complex with NADPH to 1.95-A resolution. The molecule contains two domains, a catalytic domain with a novel open twisted alpha/beta motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.     

Cross-presentation of HLA class I epitopes from exogenous NY-ESO-1 polypeptides by nonprofessional APCs

NY-ESO-1, a germ cell Ag often detected in tumor tissues, frequently elicits Ab and CD8(+) T cell responses in cancer patients. Overlapping long peptides spanning the NY-ESO-1 sequence have been used to map HLA class I-restricted epitopes recognized by NY-ESO-1-specific CD8(+) T lymphocytes. To address the antigenicity of long peptides, we analyzed two synthetic 30-mer peptides from NY-ESO-1, polypeptides 80-109 and 145-174, for their capacity to be processed by APCs and to stimulate CD8(+) T cells. By incubating APCs with polypeptides at different temperatures or in the presence of protease inhibitors, we found that NY-ESO-1 polypeptides were rapidly internalized by B cells, T2 cells, or PBLs and submitted to cellular proteolytic action to yield nonamer epitopes presented by HLA class I. Polypeptides were also immunogenic in vitro and stimulated the expansion of CD8(+) T cells against naturally processed NY-ESO-1 epitopes in the context of three different HLA class I alleles. Polypeptides can thus serve as exogenous Ags that are cross-presented on HLA class I without requiring the action of professional APCs. These findings support innovative vaccination strategies using NY-ESO-1 polypeptides that would circumvent current limitations of HLA class I peptide vaccination, i.e., HLA eligibility criteria and knowledge of epitope, while allowing for facilitated immunogenicity in the presence of helper epitopes.     

Estimation of single-trial multicomponent ERPs: Differentially variable component analysis (dVCA)

A Bayesian inference framework for estimating the parameters of single-trial, multicomponent, event-related potentials is presented. Single-trial recordings are modeled as the linear combination of ongoing activity and multicomponent waveforms that are relatively phase-locked to certain sensory or motor events. Each component is assumed to have a trial-invariant waveform with trial-dependent amplitude scaling factors and latency shifts. A Maximum a Posteriori solution of this model is implemented via an iterative algorithm from which the components waveform, single-trial amplitude scaling factors and latency shifts are estimated. Multiple components can be derived from a single-channel recording based on their differential variability, an aspect in contrast with other component analysis techniques (e.g., independent component analysis) where the number of components estimated is equal to or smaller than the number of recording channels. Furthermore, we show that, by subtracting out the estimated single-trial components from each of the single-trial recordings, one can estimate the ongoing activity, thus providing additional information concerning task-related brain dynamics. We test this approach, which we name differentially variable component analysis (dVCA), on simulated data and apply it to an experimental dataset consisting of intracortically recorded local field potentials from monkeys performing a visuomotor pattern discrimination task. AcknowledgementsThis work was supported by NIMH (MH64204 and MH42900), NSF (IBN0090717), ONR (N000149910062), T32M07288, NARSAD Young Investigator Award (KHK), NASA IDU/IS/CICT Program, and the NASA Aerospace Technology Enterprise.     

Glycophorin A somatic cell mutations in a population living in the proximity of the Semipalatinsk nuclear test site

The glycophorin A (GPA) somatic mutation assay was performed to evaluate the magnitude of exposure to ionizing radiation among the human population living in the vicinity of the Semipalatinsk nuclear test site in Kazakhstan. All together, 113 blood samples were analyzed from three generations of people living in villages that were under the trail of the radioactive cloud from the first Soviet surface nuclear test performed in August 1949 and from later tests. The oldest generation (P0) lived in the area at the time of testing, whereas the younger generations (F1, F2) were exposed to smaller doses from the residual fallout and later tests. The GPA assay did not reveal significant differences in the variant cell frequencies for all subjects selected from the Semipalatinsk area compared with 74 matched controls living in a noncontaminated area. However, a significant increase (P < 0.05) in the mean allele-loss ON variant frequency was observed among the exposed P0 generation (12 x 10(-6)) in comparison to controls (7 x 10(-6)). Considering the sensitivity of the GPA assay, the results suggest that the mean dose to the P0 generation of the affected villages was relatively low, a finding which is in accordance to the conclusions obtained from other biological assays performed on the same population.